Test if antitumor T cells use a putative super-enhancer in the Il1rl2-Il1rl1 intergenic region to form "innate-like" responses to cytokines

项目摘要 新一代基于T细胞的癌症免疫治疗，最好的例子是检查点抑制剂 抗-CTLA-4和抗-PD-1/PD-L1，可以在癌症患者中引发治疗性抗肿瘤免疫。然而，尽管如此， 这些治疗反应通常不持久或仅在一部分患者中经历，可能是由于 肿瘤采用多种免疫抑制机制来破坏抗肿瘤T细胞的作用。为 例如，表达抗原受体（TCR）的T细胞，其识别具有高表达的MHC限制性肿瘤表位。 亲合力/亲和力具有经历功能失活或缺失的倾向，因此溶细胞效应物T 位于肿瘤内的细胞（CTL）通常具有有限的识别肿瘤和被肿瘤触发的能力， 表位因此，扩增弱肿瘤表位识别，或甚至诱导TCR非依赖性T细胞增殖的策略， 肿瘤内激活，可能会增强T细胞介导的抗肿瘤功效。在这方面，我们以前发现， 用抗肿瘤促进共刺激激动剂编程的效应T细胞可以被触发， 通过TCR非依赖性过程去分化和分泌杀肿瘤细胞因子IFN-γ， 用STAT激活细胞因子（IL-2或IL-12）加IL-1家族成员（IL-33或IL-36）刺激。这 “先天样”反应类似于先天免疫细胞如NK细胞如何通过细胞因子激活而被触发。 IL-12加IL-18的组合，并可能有助于解释为什么IL-36在肿瘤中的存在 微环境限制肿瘤生长。此外，参与这种2-细胞因子触发途径将是有效的 提高癌症免疫治疗效果的方法。在分析这种2-细胞因子的机制时， 使用用IL-2 + IL-36组合处理的共刺激的CD 8 + T细胞，发现IL-2 通过快速诱导IL-36 R mRNA（由Il 1 rl 2基因编码）在细胞中表达， JAK/STAT依赖方式。染色质免疫沉淀与高通量测序（ChIP-seq） 揭示IL-2诱导STAT 5与位于整个细胞中的五个单独的STAT/GAS共有元件结合， 在Il 1 rl 2和Il 1 rl 1之间的大的~75 kb基因间区域（编码也由IL-2诱导的IL-33 R）。 引人注目的是，RNA聚合酶II在IL-2刺激之前结合相同的位点。这些结果，与一个 对从表达IFN-γ的T细胞产生的可生物学获得的ChIP-seq数据集的先前生物信息学分析 细胞，形成了新的总体假设的基础，将在本项目中进行测试，即Il 1 rl 2-Il 1 rl 1 基因间区域包含一个超级增强子（SE），作为一个分子枢纽，以促进串扰 STAT激活和IL-1家族细胞因子在“先天性”抗肿瘤T细胞应答中的作用。

Project Summary The new generation of T cell-based cancer immunotherapeutics, best exemplified by the checkpoint inhibitors anti-CTLA-4 and anti-PD-1/PD-L1, can elicit therapeutic antitumor immunity in cancer patients. Nevertheless, these therapeutic responses are often not durable or only experienced in a subset of patients, likely owing to a variety of immunosuppressive mechanisms employed by tumors to subvert the action of antitumor T cells. For instance, T cells expressing antigen receptors (TCRs) that recognize MHC restricted-tumor epitopes with high avidity/affinity have a propensity to undergo functional inactivation or deletion, and hence cytolytic effector T cells (CTL) localized within tumors generally have a limited ability to recognize and be triggered by tumor epitopes. Hence, strategies to amplify weak tumor epitope recognition, or even induce TCR-independent T cell activation intratumorally, might boost T cell-mediated antitumor efficacy. In this regard, we previously found that effector T cells programmed with antitumor promoting costimulatory agonists can be triggered to degranulate and secrete the tumoricidal cytokine IFN-γ through a TCR-independent process that involves stimulation with a STAT-activating cytokine (IL-2 or IL-12) plus an IL-1 family member (IL-33 or IL-36). This “innate-like” response is akin to how innate immune cells such as NK cells can be triggered through cytokine combinations such as IL-12 plus IL-18, and may help explain why the presence of IL-36 in the tumor microenvironment limits tumor growth. Further, engaging this 2-cytokine triggering pathway will be an effective means to boost the efficacy of cancer immunotherapies. In analyzing the mechanism of this 2-cytokine pathway using costimulated CD8+ T cells treated with the IL-2 + IL-36 combination, it was found that IL-2 primes for IL-36 responsiveness by rapidly inducing IL-36R mRNA (encoded by the Il1rl2 gene) in a JAK/STAT-dependent manner. Chromatin immunoprecipitation with high throughput sequencing (ChIP-seq) revealed that IL-2 induces STAT5 binding to five separate STAT/GAS consensus elements located throughout a large ~75 kb intergenic region between Il1rl2 and Il1rl1 (encoding IL-33R that is also induced by IL-2). Strikingly, RNA polymerase II bound the same sites prior to IL-2 stimulation. These results, in concert with a previous bioinformatic analysis of publically available ChIP-seq data sets generated from IFN-γ-expressing T cells, forms the basis of the novel overarching hypothesis that will be tested in this project that the Il1rl2-Il1rl1 intergenic region contains a super-enhancer (SE) that serves as a molecular hub to facilitate cross-talk between STAT-activating and IL-1 family cytokines during “innate-like” antitumor T cell responses.